Mechanism and Specificity of Pentachloropseudilin-mediated Inhibition of Myosin Motor Activity

Chinthalapudi, Krishna; Taft, Manuel H.; Martin, René; Heissler, Sarah M.; Preller, Matthias; Hartmann, Falk K.; Brandstaetter, Hemma; Kendrick‐Jones, John; Tsiavaliaris, Georgios; Gutzeit, Herwig O.; Fedorov, Roman; Buß, Folma; Knölker, Hans‐Joachim; Coluccio, Lynne M.; Manstein, Dietmar J.

doi:10.1074/jbc.m111.239210

Cited by 60 publications

(88 citation statements)

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“…It is important to note that although the potent inhibition of myosin I proteins by PCIP has been characterized in greatest detail, the drug also inhibits the ATPase activity of myosin Vb and nonmuscle myosin II at higher concentrations (IC 50 >90 μM) [54]. This lack of specificity at higher concentrations must be taken into account when designing in vivo experiments using PCIP.…”

Section: Small-molecule Inhibitors Of Myosinsmentioning

confidence: 99%

“…Structural studies of the binding of PCIP to a model myosin (Dictyostelium discoideum myosin II) indicate that the compound binds to an allosteric pocket in the myosin motor domain and thus acts as a noncompetitive inhibitor of motor activity [54]. The PCIP-binding pocket is located near actin-binding residues at the tip of the 50-kDa cleft in the myosin motor domain, approximately 16 Å from the nucleotide binding site and 7.5 Å from the hydrophobic pocket bound by blebbistatin.…”

Section: Small-molecule Inhibitors Of Myosin Imentioning

confidence: 99%

“…Pentachloropseudilin-The small molecule pentachloropseudilin (PCIP) has been identified as an inhibitor of myosin I function ( [54].…”

Section: Small-molecule Inhibitors Of Myosin Imentioning

confidence: 99%

“…PCIP binding to this allosteric pocket reduces ATPase activity via a conserved communication pathway between the allosteric and nucleotide binding sites that results in local structural changes in the myosin, reduction of coupling between the actin and nucleotide binding sites, and global changes in myosin dynamics (Figure 1) [54,55]. Complementary computational studies indicate that the specific preference of PCIP for the binding pocket in class I myosins may stem from the presence of a relatively higher number of polar/charged residues within the binding site of this myosin class [55].It is important to note that although the potent inhibition of myosin I proteins by PCIP has been characterized in greatest detail, the drug also inhibits the ATPase activity of myosin Vb and nonmuscle myosin II at higher concentrations (IC 50 >90 μM) [54]. This lack of specificity at higher concentrations must be taken into account when designing in vivo experiments using PCIP.…”

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Small-Molecule Inhibitors of Myosin Proteins

et al. 2012

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Advances in screening and computational methods have enhanced recent efforts to discover/ design small-molecule protein inhibitors. One attractive target for inhibition is the myosin family of motor proteins. Myosins function in a wide variety of cellular processes, from intracellular trafficking to cell motility, and are implicated in several human diseases (e.g., cancer, hypertrophic cardiomyopathy, deafness and many neurological disorders). Potent and selective myosin inhibitors are, therefore, not only a tool for understanding myosin function, but are also a resource for developing treatments for diseases involving myosin dysfunction or overactivity. This review will provide a brief overview of the characteristics and scientific/therapeutic applications of the presently identified small-molecule myosin inhibitors before discussing the future of myosin inhibitor and activator design.The identification and characterization of pharmacological compounds that inhibit the functional activity of one or more specific proteins or processes has been the subject of much scientific investigation. On a basic science level, these membrane-permeable compounds provide the scientific community with a tool for the targeted and functional inhibition of a given protein in the cell; a potent means of evaluating the intracellular functions of that protein [1,2]. From a biomedical standpoint, the characterization of these small-molecule inhibitors affords an opportunity for the development of novel disease treatments centering on the repression of an offensive molecule or the reversal of its downstream effects [3][4][5].At present, several complementary methods for obtaining suitable small-molecule inhibitors of specific proteins exist. Traditional methods in inhibitor discovery involve the systematic testing of a series of chemically synthesized or naturally occurring compounds. Advances in robotics and data processing have made it possible to use high-throughput screens to test libraries of thousands or even millions of potential drugs for their ability to inhibit the function of a specific protein in a targeted biochemical or cellular assay [6][7][8]. These inhibitor discovery processes are complemented by more precise methods in small-molecule inhibitor design. Structure-based methods rely on the use of x-ray crystallographic or NMRbased structures of a protein of interest to design small molecules likely to bind and inhibit protein function [9,10]. Computer-aided inhibitor design uses computational methods to optimize potential inhibitors identified by screening or structure-based methods, to virtually © 2013 Folma Buss * Author for correspondence: Tel.: +44 1223 763348, Fax: +44 1223 762640, fb207@cam.ac.uk. Financial & competing interests disclosureThe authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance w...

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Section: Small-molecule Inhibitors Of Myosinsmentioning

confidence: 99%

Section: Small-molecule Inhibitors Of Myosin Imentioning

confidence: 99%

“…Pentachloropseudilin-The small molecule pentachloropseudilin (PCIP) has been identified as an inhibitor of myosin I function ( [54].…”

Section: Small-molecule Inhibitors Of Myosin Imentioning

confidence: 99%

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Small-Molecule Inhibitors of Myosin Proteins

et al. 2012

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“…As BDM has turned out to have a broad effect on many other proteins (7) and the inhibitory effect of BTS is limited to fast skeletal muscle myosin, until now blebbistatin has been the only potent tool for specific blocking of myosin II-dependent processes in various species and cell types. Recent data have shown that halogenated pseudilins also have nonspecific inhibitory effects on myosin II isoforms (8)(9)(10).…”

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Azidoblebbistatin, a photoreactive myosin inhibitor

Képiró¹,

Várkuti²,

Bodor³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Photoreactive compounds are important tools in life sciences that allow precisely timed covalent crosslinking of ligands and targets. Using a unique technique we have synthesized azidoblebbistatin, which is a derivative of blebbistatin, the most widely used myosin inhibitor. Without UV irradiation azidoblebbistatin exhibits identical inhibitory properties to those of blebbistatin. Using UV irradiation, azidoblebbistatin can be covalently crosslinked to myosin, which greatly enhances its in vitro and in vivo effectiveness. Photo-crosslinking also eliminates limitations associated with the relatively low myosin affinity and water solubility of blebbistatin. The wavelength used for photo-crosslinking is not toxic for cells and tissues, which confers a great advantage in in vivo tests. Because the crosslink results in an irreversible association of the inhibitor to myosin and the irradiation eliminates the residual activity of unbound inhibitor molecules, azidoblebbistatin has a great potential to become a highly effective tool in both structural studies of actomyosin contractility and the investigation of cellular and physiological functions of myosin II. We used azidoblebbistatin to identify previously unknown low-affinity targets of the inhibitor (EC 50 ≥ 50 μM) in Dictyostelium discoideum, while the strongest interactant was found to be myosin II (EC 50 = 5 μM). Our results demonstrate that azidoblebbistatin, and potentially other azidated drugs, can become highly useful tools for the identification of strong-and weak-binding cellular targets and the determination of the apparent binding affinities in in vivo conditions. interactom profile | azidation | photoactivation V arious biological processes, including muscle contraction, cell migration, differentiation, and cytokinesis require the activity of myosin II, a class of actin-based ATP-driven motor proteins. Cell-permeable small molecules that can perturb myosin functions have greatly aided the dissection and understanding of molecular processes underlying the above phenomena. Myosin II inhibitors described to date include 2,3-butanedione monoxime (BDM) (1, 2), N-benzyl-p-toluenesulphonamide (BTS) (3), and blebbistatin (4-6). As BDM has turned out to have a broad effect on many other proteins (7) and the inhibitory effect of BTS is limited to fast skeletal muscle myosin, until now blebbistatin has been the only potent tool for specific blocking of myosin II-dependent processes in various species and cell types. Recent data have shown that halogenated pseudilins also have nonspecific inhibitory effects on myosin II isoforms (8-10).In vitro, blebbistatin inhibits vertebrate striated muscle and nonmuscle myosin II isoforms as well as Dictyostelium discoideum (Dd) myosin II by about 95%, with an IC 50 of 0.5-5 μM (11). Vertebrate smooth muscle and Acanthamoeba myosin II are incompletely inhibited even at high blebbistatin concentrations. In vivo experiments performed with Dd showed that the effective inhibition of myosin II-dependent processes, including growth in...

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Pentachloropseudilin Inhibits Transforming Growth Factor‐β (TGF‐β) Activity by Accelerating Cell‐Surface Type II TGF‐β Receptor Turnover in Target Cells

et al. 2018

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Pentachloropseudilin (PClP) is a chlorinated phenylpyrrole compound that was first isolated from Actinoplanes (ATCC33002), and its structure has been confirmed by chemical synthesis. PClP shows broad antimicrobial activity against Gram-negative and Gram-positive bacteria, protozoa, fungi, and yeast. In mammalian cells, PClP is known to act as a reversible and allosteric inhibitor of myosin 1c (Myo1c). Herein, we report that PCIP is a potent inhibitor of transforming growth factor-β (TGF-β)-stimulated signaling. PCIP inhibits TGF-β-stimulated Smad2/3 phosphorylation and plasminogen activator inhibitor-1 (PAI-1) promoter activation with an IC of 0.1 μm in target cells (A549, HepG2, and Mv1Lu cells). In addition, PCIP attenuates TGF-β-stimulated expression of vimentin, N-cadherin, and fibronectin and, thus, blocks TGF-β-induced epithelial to mesenchymal transition (EMT) in these cells. Furthermore, cell-surface labeling and immunoblot analysis indicates that PCIP suppresses TGF-β-stimulated cellular responses by attenuating cell-surface expression of the type II TGF-β receptor through accelerating caveolae-mediated internalization followed by primarily lysosome-dependent degradation of the receptor, as demonstrated by sucrose density gradient analysis and immune fluorescence staining.

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Mechanism and Specificity of Pentachloropseudilin-mediated Inhibition of Myosin Motor Activity

Cited by 60 publications

References 26 publications

Small-Molecule Inhibitors of Myosin Proteins

Small-Molecule Inhibitors of Myosin Proteins

Azidoblebbistatin, a photoreactive myosin inhibitor

Pentachloropseudilin Inhibits Transforming Growth Factor‐β (TGF‐β) Activity by Accelerating Cell‐Surface Type II TGF‐β Receptor Turnover in Target Cells

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